Radio et al. Parasites & Vectors (2018) 11:56 DOI 10.1186/s13071-017-2553-2

RESEARCH Open Access Pleiotropic alterations in expression in Latin American Fasciola hepatica isolates with different susceptibility to drugs Santiago Radio1,2, Santiago Fontenla1, Victoria Solana3, Anna C. Matos Salim4, Flávio Marcos Gomes Araújo4, Pedro Ortiz5, Cristian Hoban5, Estefan Miranda6, Valeria Gayo7, Fabiano Sviatopolk-Mirsky Pais4, Hugo Solana3, Guilherme Oliveira4,8, Pablo Smircich1,2,9* and José F. Tort1*

Abstract Background: Fasciola hepatica is the main agent of fasciolosis, a zoonotic disease affecting livestock worldwide, and an emerging food-borne disease in humans. Even when effective treatments are available, drugs are costly and can result in tolerance, liver damage and normally they do not prevent reinfection. Drug-resistant strains in livestock have been reported in various countries and, more worryingly, drug resistance in human cases has emerged in South America. The present study aims to characterize the transcriptome of two South American resistant isolates, the Cajamarca isolate from Peru, resistant to both triclabendazole and albendazole (TCBZR/ABZR) and the Rubino isolate from Uruguay, resistant to ABZ (TCBZS/ABZR), and compare them to a sensitive strain (Cenapa, Mexico, TCBZS/ABZS) to reveal putative molecular mechanisms leading to drug resistance. Results: We observed a major reduction in transcription in the Cajamarca TCBZR/ABZR isolate in comparison to the other isolates. While most of the differentially expressed are still unannotated, several trends could be detected. Specific reduction in the expression levels of cytoskeleton proteins was consistent with a role of tubulins as putative targets of triclabendazole (TCBZ). A marked reduction of adenylate might be underlying pleiotropic effects on diverse metabolic pathways of the parasite. Upregulation of GST mu isoforms suggests this detoxifying mechanism as one of the strategies associated with resistance. Conclusions: Our results stress the value of transcriptomic approaches as a means of providing novel insights to advance the understanding of drug mode of action and drug resistance. The results provide evidence for pleiotropic variations in drug-resistant isolates consistent with early observations of TCBZ and ABZ effects and recent proteomic findings. Keywords: Fascola hepatica, Drug resistance, American isolates, Triclabendazole, Albendazole, Transcriptomics

Background issue in human health, affecting roughly 2.6 million people Fasciolosis is indisputably one of the most widely distrib- worldwide. For this reason it has been considered as a re- uted zoonotic diseases, affecting no less than 300 million emerging neglected disease by the WHO [3]. In the cattle and 250 million sheep worldwide. The economical Americas the disease is widespread in livestock and is an cost of the disease has been valued at 3 billion dollars annu- important human food-borne infection in the Altiplano ally [1, 2]. This huge economic impact from direct losses region of Bolivia and Peru [4]. might be an underestimate considering indirect costs of Although effective treatments are available, drugs are treatment, or loss of animal workforce in less industrialized costly and usually do not block reinfection. Liver fluke countries. Furthermore, fasciolosis is emerging as a relevant drug resistance is a preoccupying productive problem in Latin America and a concern in medicine since human cases have emerged [4]. Triclabendazole (TCBZ) treat- * Correspondence: [email protected]; [email protected] 1Departamento de Genética, Facultad de Medicina, Universidad de la ment failure has been reported in Brazil [5], Argentina [6] Republica, UDELAR, Montevideo, Uruguay and Peru [7–9]. Moreover, resistance to albendazole Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Radio et al. Parasites & Vectors (2018) 11:56 Page 2 of 11

(ABZ) has been reported in Argentina [10], Uruguay [11], vesicle traffic, alterations in reproductive tissues and Chile and Bolivia [5]. More worryingly, ABZ resistance vitelline cells, and reduction in egg production [33, 34]. accompanied by reduced effectivity of TCBZ has been However, despite these similarities, differences in the registered in Bolivia [12] and Peru [13], and resistance to metabolism of worms treated with these drugs are both drugs has been described in an isolate from the suggestive of diverse targets or mechanisms. Cajamarca valley of Peru [9, 11]. This phenomenon of It has been shown that the metabolism of TCBZ to double resistance compromises the idea of combined drug triclabendazole sulphoxide (TCBZ.SO) and TCBZ.SO to treatments. More recently, TCBZ resistance in humans triclabendazole sulphone (TCBZ.SO2) is greater in has been reported in Chile and Peru [14, 15]. There is a TCBZ-R than in TCBZ-S isolates [35–37]. Interestingly, pressing need to understand epidemiological and mechan- the uptake of TCBZ and TCBZ.SO by TCBZ-R fluke istic aspects of drug resistance emergence. isolates is significantly lower than in TCBZ-S flukes, while Despite being in use for more than 30 years, the exact theuptakeofABZissimilarinbothstrains[35,38].Theef- mechanism of action of TCBZ is still not completely fect can be reversed by incubating the TCBZ-R flukes in elucidated [16]. One of the earliest and more complete the presence of ivermectin, a substrate of P-glycoprotein biochemical studies provides early evidence of (PGP) drug efflux pump. While disruption of the tegument pleiotropic effects [17]. The study documented that the is markedly reduced in TCBZ-R flukes, the co-incubation drug was absorbed through the tegument affecting the with R(+)-verapamil, another PGP inhibitor, gives rise to motility of the parasite in a dose-dependent manner. severe tegumental lesions [39, 40], highlighting PGP as one This effect was associated with changes in the worm’s of the possible detoxifying mechanisms. A similar effect of resting tegument membrane potentials. In addition, an increased tegument disruptionisseenwhenTCBZ-Rflukes effect on tubulin binding was also observed. Further- are incubated with methimazole, an inhibitor of the flavin more, an anaerobic metabolic stimulation was suggested mono-oxigenases (FMO) [41]. Ketoconazole, an inhibitor by the increase in propionate and acetate production of CYP450 [42, 43] also produce as similar phenotype, sug- without affecting ATP levels. A general reduction of gesting that these pathways might be associated with drug secreted proteases was also described [17], an effect that resistance as well. It was hypothesized that some of these might be associated with a reported general inhibition of might be upregulated in the resistant strains [35, protein synthesis [18]. Several studies have focused on 36]. Consistent with this, an increased activity of tegument disruption, one of the major effects of drug detoxifying enzymes gluthatione S- (GST), treatment [19–24]. This was generally associated with a carboxyl esterase and carbonyl reductase, was observed in putative role of tubulin as target, based on similarities to TCBZ-treated worms [44], and a higher GST response was findings on other benzimidazole effects against nema- observedintheTCBZ-RSligostrainincomparisontothe todes [16, 25]. The ability to compete with colchicine, a Cullompton sensitive isolate [45]. A comparative proteomic known inhibitor of tubulin polymerization, was an early study of the Sligo and Cullompton isolates showed test for tubulin binding [17, 26]. The role of microtu- variation in energy metabolic enzymes, detoxifying enzymes bules in TCBZ pathogenesis was further suggested by and structural proteins, confirming the pleiotropic nature several lines of evidence, like the detection of cell of TCBZ effects, and reinforced suggestions of differential division inhibition in vitelline and reproductive cells expression of several proteins [46]. [27–29], the reduction in the transport of tegumental It is therefore clear that anthelmintic drugs produce com- secretory bodies [22], and the inhibition of tubulin plex effects on the liver fluke, affecting the metabolism, immunostaining [30, 31]. Surprisingly, TCBZ was recently physiology and morphology of the parasite. Drug-resistant reported to inhibit adenylate cyclase activity in yeast, isolates seem to utilize diverse detoxifying mechanisms to activating the stress response [32]. This effect has not been cope with these drugs [47]. We sought to shed new light observed in the liver fluke so far, but considering the role of into drug resistance by using a genomics approach, which cAMP as second messenger, it might provide an explan- has been demonstrated to be very powerful in schistosomes ation for the pleiotropic effects of the drug. [48–52]. Similar efforts have been initiated in F. hepatica Interestingly, TCBZ is quite specific for fasciolids, [53], starting from well characterized European TCBZ-R being ineffective against nematodes. On the other hand, and TCBZ-S isolates and their genome sequences [54, 55]. out of the benzimidazolic drugs used for gastrointestinal In this work we add a transcriptomic perspective to the roundworms, only ABZ is effective against the adult current knowledge via an in depth analysis of the basal stage of Fasciola spp. [33]. These differences and the fact transcriptomic state of three isolates from the Americas that ABZ is usually effective against TCBZ-resistant with different susceptibilities to TCBZ and ABZ. This isolates suggest that different mechanisms might be report would set the baseline for upcoming comparative underlying the effect of each benzimidazolic drug. ABZ studies on variations of gene expression upon exposure to also induces tegument damage, disruption of tegumental the different anthelminthic drugs. Radio et al. Parasites & Vectors (2018) 11:56 Page 3 of 11

Methods established by computing pairwise Pearson’s correlation Strains coefficients in R. Differentially expressed genes were defined Three Fasciola hepatica isolates with different susceptibil- using DESeq2 (using the Wald Test implemented in the ities to triclabendazole (TCBZ) and albendazole (ABZ) were package with 4 degrees of freedom) from pairwise compari- analyzed. The “Cajamarca” isolate was originally obtained by sons of the log2-transformed normalized expression Dr Pedro Ortiz from infected cattle in Cajamarca, Peru. It estimates, establishing a minimum fold change of 2 and a has been maintained for five years in sheep and character- false discovery rate (FDR) (controlled using the Benjamini & ized in their laboratory as resistant to both ABZ and TCBZ Hochberg’s method [61]) corrected P-value lower than 0.05. [9, 11]. The “Rubino” isolate (originally obtained from cattle in Salto, Uruguay) is resistant to ABZ but sensitive to TCBZ [11]. It has been maintained in sheep for eight years by Dr Functional enrichment analysis Valeria Gayo in the DILAVE “Miguel C. Rubino”.Theiso- Lists of DE genes were analyzed with the TopGO late is routinely used to test formulations of Closantel and Biocondutor package in R [62] to assess enriched gene TCBZ, since it is sensitive to both. Similarly, “Cenapa” is an ontology categories (using Fisher’s exact test implemented isolate sensitive to both drugs that has been maintained for in the package with 2 degrees of freedom; a P-value < 0.01 more than a decade in sheep, and is routinely used by the was considered significant). The parameters orderBy veterinary health authorities of the Mexican government to = “classicFisher” and ranksOf = “classicFisher” were set for evaluate the efficacy of anthelminthics. The Cenapa isolate visualization. GO annotation was retrieved from Wormbase was kindly provided by Dr Estefan Miranda. The three Parasite (parasite.wormbase.org) [63]. laboratories followed protocols approved by the respective local Committees of Animal Experimentation, in accordance totherecommendationsofGuidefortheCareandUseof SNP calling Laboratory Animals [56]. All isolates are maintained in SNPs were called from RNAseq mappings to the reference sheep without selective drug pressure. genome using mpileup (-uf parameters) and bcftools (-mv parameters) from samtools [64]. Putative phenotypic effects RNA-sequencing and pre-processing of the reads of the variants called were assessed with the variant effect Adult flukes were obtained from infected sheep livers and predictor script (part of the Ensembl tools [65]. Synonymous stored immediately in RNAlater. No macroscopic mor- codon variants and other low impact mutations were not phological differences were observed between flukes. included in the analysis. PolyA+ RNA was purified from single adult worms of the different isolates in duplicates and used to generate paired end (PE) libraries using the TrueSeq LT kit (Illumina, San KEGG orthology annotation Diego, USA). Samples were sequenced in the Illumina The GhostKOALA tool [66] was used to annotate the Platform at the CPqRR sequencing facilities at FIOCRUZ, predicted proteome. Genes belonging to common Belo Horizonte, to obtain 117 million 76 bp pair end housekeeping functions were identified using the KEGG reads. The resulting sequences were quality trimmed and Brite reconstruction list [67]. Selected categories, gene mapped to the F. hepatica reference genome (WormBase IDs and genome annotation are shown in Additional Parasite Acc: PRJEB6687) [54] using CLC Genomics files as indicated in the text. Workbench v7 (Qiagen, Aarhus C, Denmark) with default parameters. A good coverage of predicted genes (over 80%) was observed. A summary of the number of reads Results and discussion obtained in each step is shown in Additional file 1: Table A lower transcription level is observed for a significant S1. Raw sequencing data were submitted to SRA under amount of Cajamarca genes accession PRJNA339158. To obtain a global picture of the differences of the transcrip- tomic profiles of the different isolates, samples were clus- Differential expression (DE) determination tered and pairwise correlations were computed. Figure 1a Differential expression was analyzed using different tools of shows that while duplicates were very consistent, the the Bioconductor suite of bioinformatics packages [57, 58]. Cajamarca isolate displayed the lowest level of correlation to To obtain expression estimates, mapped reads were theothersamples.Thisdifferenceismostlyexplainedby counted for each gene using the summarizeOverlaps comparatively low mRNA steady state levels of many function from the GenomicAlingments package [59] and transcriptsinthisstrain(Fig.1b and Additional file 2: Figure log2-transformed. To account for sequencing depth in S1a). On the other hand, differences in the gene expression differential expression analysis, raw read counts were were subtler between the Rubino and Cenapa samples normalized with DESeq2 [60]. Replicate consistency was (Additional file 2: Figure S1b). Radio et al. Parasites & Vectors (2018) 11:56 Page 4 of 11

Fig. 1 Overview of sample distances and differential expression. a Heatmap of the Euclidean sample to sample distances shown in a white to blue scale (blue represents higher correlation). Both replicates for each sample are shown, with the corresponding clustering dendrograms on the sides. b Volcano plot showing the differential expression between the Cajamarca and Rubino transcripts. Red dots represent differentially expressed genes (log2 fold change > 2, P-value < 0.01)

Housekeeping gene expression is not biased between Notably, early studies of the effect of TCBZ reported a isolates marked drop in protein synthesis [18], consistent with SincewesawareductionoftranscriptionintheTCBZR/ morphological observations of changes in heterochromatin, ABZR Cajamarca isolate in relation to the other two iso- the disappearance of the nucleolus and the subsequent lates, we wanted to check if the asymmetrical distribution reduction of ribosomes, reduction in Golgi complexes, and of differentially expressed genes (DEG) among the strains secretory bodies in tegumental cells [22]. Therefore, it is was due to a general lower transcript level in the Cajamarca tempting to consider that the study of differentially isolate that normalization was not able to account for. expressed (DE) genes might help to pinpoint some possible Interestingly, we detected that housekeeping genes (such as candidates involved in the resistance phenotype. aminoacyl tRNA synthetases, DNA polymerase subunits, proteasome subunits and spliceosome subunits) do not The top differentially expressed genes are mostly not show differences of expression among any of the strains annotated (Fig. 2 and Additional file 3: Figure S2). These results Pairwise comparisons were performed between the isolates indicate that Cajamarca samples are not globally skewed using DESeq2 package. Almost half of the downregulated toward less expression, but rather different gene families or upregulated genes in each pairwise list are currently might be differentially affected. These results are puzzling devoid of any annotation. We focused on the top 20 since no drug selective pressure was applied to these upregulated or downregulated DEG in each pairwise worms, and it might reflect a basal status of the isolate. comparison (Additional file 4: Table S2). Even within this selected set, 41 out of 58 of the downregulated DEG are of unknown function, and diverse functions are present in the remaining annotated genes. A similar scenario was observed for the upregulated DEG where 37 of 47 were unannotated, highlighting the still incomplete nature of the available genome annotation. However, 3 out of 10 of the annotated DEGs upregulated in the Cajamarca and Rubino resistant strains, contain putative CUB domains (IPR000859). This domain (for Complement C1r/C1s, Uegf and Bmp1) comprises more than 100 amino acids and is usually found in the extracellular- or plasma membrane- associated proteins with diverse functions. Several mamma- lian CUB containing proteins are proteases with calcium binding EGF domains, taking part in pleiotropic functions Fig. 2 Correlation of housekeeping gene expression. Scatterplot like complement activation, developmental patterning, showing the correlation of Rubino normalized counts per gene versus neurotransmission and cell signaling [68]. CUB domain their Cajamarca counterparts. Filled circles highlight the expression of containing proteins found in the F. hepatica genome are genes belonging to housekeeping functions: dark cyan, aminoacyl tRNA generally short with no other associated domains, but this synthetases; dark green, core DNA polymerase subunits; dark magenta, might well be a consequence of the still fragmented nature core proteasome subunits; light green, core spliceosome subunits of the assembly. Interestingly, upregulation of CUB domain Radio et al. Parasites & Vectors (2018) 11:56 Page 5 of 11

containing proteins has been observed in C. elegans in putative role of tubulins as targets of ABZ, TCBZ and other response to albendazole administration [69]. Although little benzimidazole-based drugs [70, 71]. is known of the relevance or function of these proteins in These results prompted us to further characterize the helminths, the upregulation in the resistant isolates even expression of cytoskeleton-related gene families. Figure 4 without exposure to the drug is noteworthy. shows the expression profile of the α and β tubulin gene families (Fig. 4a, b) and motor protein families (kinesins Cytoskeleton related genes are less expressed in the and dyneins Fig. 4c, d). In all cases the Cajamarca isolate Cajamarca strain showed lower levels of gene expression for these families The entire lists of DEG in each pairwise comparison were when compared to the other strains, which were not sig- subjected to GO functional category enrichment analysis. nificantly different among them (Student’s t-test P <0.05, Remarkably, several terms related to cytoskeleton structure see legend of Fig. 4 for exact P-values of each compari- and function showed a significant enrichment in the list of son). In particular, α-tubulin and β-tubulin mRNAs downregulated genes in the resistant strains, especially in showed differential expression between the strains being the Cajamarca isolate (Fig. 3 and Additional file 5: Table S3). down-represented in the Cajamarca isolate (Fig. 4a, b and This is an interesting observation taking into account the Additional file 6: Table S4). Finding a skewed expression

Fig. 3 (GO) enrichment of Cajamarca downregulated genes. Directed acyclic graph showing cellular component GO categories present in the downregulated genes when comparing the Cajamarca with the Cenapa samples. Colors indicate category overrepresentation, with red being most significant. Text lines indicate GO number, category description, Fisher’s P-value and the number of genes in the set over the total number of genes in the genome for the GO category Radio et al. Parasites & Vectors (2018) 11:56 Page 6 of 11

Fig. 4 Expression levels of cytoskeleton related gene families. Boxplot showing the expression levels for mRNAs coding for α-tubulins (a), β-tubulins (b),

kinesins (c) and dyneins (d) in each sample. Samples were compared using a two-sided Student’s t-test in R. For α-tubulins CA vs RU: t(30) = -3.586, P < 0.001; CA vs CE: t(33) = -2.148, P = 0.039; RU vs CE: t(35) = 1.515, P = 0.139. For β-tubulins CA vs RU: t(15) = -2.529, P = 0.023; CA vs CE: t(16) = -1.372, P = 0.189; RU vs CE: t(16) = 1.162, P = 0.262. For kinesins CA vs RU: t(64) = -5.092, P < 0.001; CA vs CE: t(67) = -3.522, P < 0.001; RU vs CE: t(73) = 1.608, P = 0.112. For dyneins CA vs RU: t(347) = -2.473, P = 0.014; CA vs CE: t(343) = -0.207, P = 0.836; RU vs CE: t(337) = 2.407, P = 0.017. Significant comparisons are marked with asterisks (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). Abbreviations: CA, Cajamarca; RU, Rubino; CE, Cenapa; ns, not significant of tubulins and other cytoskeleton genes in a double expression has been observed in H. contortus drug- resistant isolate reinforces the notion of tubulin as a resistant strains [75, 76]. However, a previous study putative TCBZ target. failed to detect differences in transcription levels of SNPs in the β-tubulin genes have been reported as diverse β-tubulins between the TCBZ resistant Oberon molecular mechanisms of resistance development in and the Leon TCBZ sensitive isolates [77]. While these nematodes, and in particular three amino acid substitu- contradictory observations might be pointing to different tions in the Haemonchus contortus β-tubulin have been mechanisms of resistance in different strains, the associated with resistance [72, 73]. Initial studies observation that other motor proteins mRNA levels are attempting to find the same variations in F. hepatica reduced (Fig. 4c, d) clearly relates to the previous failed to find association between these changes and finding and further associates the resistance phenotype resistant status [30, 74]. Despite this, a strongly reduced with microtubule cytoskeleton function. tegument damage and little disruption of tubulin immu- Interestingly, collagen coding mRNAs were under- nostaining were observed in TCBZ resistant flukes in expressed in the Rubino isolate compared to the other comparison to a sensitive isolate [30]. Similarly, disrup- strains. The difference is particularly significant with the tion of tegument and reduction of tubulin immunostain- Cajamarca isolate where an average 5-fold transcription ing were also observed upon exposure to albendazole level was detected for collagen (Additional file 7: Table S5). sulphoxide (ABZ-SO) [34] highlighting tubulin as one of the putative targets in F. hepatica. Our results support Only a few genes associate with detoxifying pathways are the association of these proteins with the resistance differentially expressed phenomenon, but in our case, mRNA level variations Since several putative candidates genes involved in drug and not SNPs were revealed as the putative molecular resistance have been advanced [47, 78], we verified their mechanism. Notably, a similar reduction in tubulin differential expression in our three isolates. One of the Radio et al. Parasites & Vectors (2018) 11:56 Page 7 of 11

first mechanisms proposed in detoxification is the oxida- Table S7). It has been hypothesized that ABC transporters tion of the incoming drug by proteins involved in redox might be upregulated in drug-resistant strains [79]. Indeed, activity (defined by annotation of functional domains) an ABC transporter-like protein (BN1106_s3396B000087) (Additional file 8: Table S6). We detected downregula- was upregulated in the TCBZ resistant isolate tion of several redox proteins in the Cajamarca strain (Additional file 9: Table S7). Whether this is further when compared with the other two isolates, and we upregulated upon drug exposure still needs to be addressed. observed extreme variations. Anotherproposedmechanismfordrugresistanceisthe Adenylate cyclase is reduced in TCBZR isolate conjugation of the primary metabolite to proteins, such as Recently it was reported that TCBZ can inhibit adenylate the glutathione S-transferase family (GST), and other cyclase (AC) activity in yeast [32], but despite being widely detoxification enzymes. The expression pattern of these studied in F. hepatica [80], there are still no in vitro studies candidates was analyzed, and no significant differences were of AC variation in response to drug administration. To gain observed for the complete set when a FC > 2 cut-off was insight into this possible mechanism, we investigated if AC applied. However, while there is no global trend when all expression was skewed in our samples. Surprisingly we enzymes are considered, it is interesting to point out that observed a significant variation on the expression levels of some GST genes, particularly of the mu type, do have sta- several AC isoforms in both the resistant isolates in relation tistically significant variation and a modest upregulation in to the Cenapa sensitive strain. Notably, all isoforms tend to the resistant strains (FC > 2, see Additional file 9: Table S7). have consistently low transcription levels in the Cajamarca This is consistent with previous biochemical studies that isolate with some of them being significant, while variations have shown increased GST activity in the Sligo resistant in both directions were found in the ABZR Rubino isolate strain when compared to the Cullampton sensitive strain (Table 1). [44, 45]. Also an increase in GST mu was observed in the The described inhibition of AC upon exposure to only comparative proteomic study available so far [46]. TCBZ in yeast would not necessarily reduce their tran- Among the detoxification proteins, some mRNAs were scription levels. Considering the central role of AC in significantly different between the strains (Additional file 9: metabolism, its downregulation in the Cajamarca isolate

Table 1 Pairwise comparisons for all annotated adenylate cyclase genes Gene Cajamarca vs Cenapa Rubino vs Cenapa Cajamarca vs Rubino Genome annotation Kegg annotation Fold change (log2) FDR Fold change (log2) FDR Fold change (log2) FDR BN1106_s820B000180 -2.54 1.00e-06 0.78 1.49e-01 -3.32 1.42e-11 class-3/4/ K08049 ADCY9 guanylyl cyclase (Nucleotide cyclase) BN1106_s5842B000024 -2.42 4.10e-03 1.78 3.80e-03 -4.20 7.78e-09 Adenylyl cyclase class-3/4/ K08049 ADCY9 guanylyl cyclase (Nucleotide cyclase) BN1106_s1582B000143 -1.81 1.26e-04 1.24 5.50e-03 -3.05 9.42e-13 Adenylyl cyclase class-3/4/ – guanylyl cyclase (Nucleotide cyclase) BN1106_s3088B000130 -1.74 3.43e-02 1.36 5.27e-02 -3.10 6.20e-06 Adenylyl cyclase class-3/4/ – guanylyl cyclase (Nucleotide cyclase) BN1106_s2758B000091 -1.61 1.20e-02 1.67 2.09e-03 -3.28 8.90e-10 Adenylyl cyclase class-3/4/ K08041 ADCY1 guanylyl cyclase (Nucleotide cyclase) BN1106_s795B000308 -1.05 7.29e-02 -1.91 2.65e-04 0.85 2.40e-01 Adenylyl cyclase class-3/4/ K08049 ADCY9 guanylyl cyclase (Nucleotide cyclase) BN1106_s451B000364 -0.58 6.54e-01 -1.98 2.50e-02 1.40 2.07e-01 Adenylyl cyclase class-3/4/ K08049 ADCY9 guanylyl cyclase (Nucleotide cyclase) BN1106_s4307B000027 -0.48 na -0.61 na 0.00 na na K08049 ADCY9 BN1106_s1588B000215 -0.12 na -1.02 na 0.91 6.46e-01 Adenylyl cyclase class-3/4/ K08049 ADCY9 guanylyl cyclase (Nucleotide cyclase) BN1106_s1588B000216 0.13 9.35e-01 -0.78 4.20e-01 0.91 4.60e-01 Adenylyl cyclase class-3/4/ K08049 ADCY9 guanylyl cyclase (Nucleotide cyclase) IDs of DEG genes [FC > 2 or < -2 (log2 FC >1 or < -1) and an FDR < 0.05] in any comparison are highlighted in bold. The log2FC value is highlighted in italics and underlined for the upregulated genes and in bold and double underlined for the downregulated ones Radio et al. Parasites & Vectors (2018) 11:56 Page 8 of 11

may account for the pleiotropic reduction in gene ondiversemechanismsortargets.Thishighlightstheneed expression observed. Since cyclic AMP is a relevant second for studying diverse isolates in order to gain a better messenger, we investigated the expression levels of the understanding of drug action and parasite resistance main mediators A (PKA) and the RAP mechanisms. The results provided by this work are a step in guanine nucleotide exchange factors. Both genes showed this direction that we hope will impact future methods for no alterations in their transcription profile. Nevertheless, parasite control. the reduction in AC might result in a concomitant AMPc drop, which in turn would activate diverse stress responses Additional files through all these effectors without altering their transcrip- tion levels. This might provide an explanation for the Additional file 1: Table S1. General data processing overview. (XLSX 10 kb) pleiotropic effects on motility, cytoskeleton, carbohydrate Additional file 2: Figure S1. Differential expression between isolate metabolism, and activation ofstressanddetoxifying pairs. Volcano plot showing the differential expression of transcripts between Cajamarca and Cenapa (a) and between Rubino and Cenapa enzymes. In any case, carefully controlled in vitro (b). Red dots represent differentially expressed genes (log2 fold change experiments of TCBZ inhibition of AC activity in flukes are >2,P-value < 0.01). (TIFF 1086 kb) needed to confirm these hypotheses. Additional file 3: Figure S2. a Correlation of normalized counts and Interestingly, our results are generally consistent with the housekeeping genes. Scatterplot showing the correlation of Cenapa normalized counts per gene versus their Cajamarca counterparts. b Scatterplot comparative proteomics results that found variations in showing the correlation of Cenapa normalized counts per gene versus their several metabolic enzymes, as well as in stress response Rubino counterparts. Full circles highlight the expression of genes belonging proteins and structural proteins [46]. Several of the proteins to housekeeping functions. Colors are as in Fig. 2. (TIFF 1042 kb) and genes found to be differentially expressed at transcript Additional file 4: Table S2. Top 20 upregulated (a) and downregulated (b) genes for each pairwise comparison. (XLSX 16 kb) level in this study (Additional file 10: Table S8) were also Additional file 5: Table S3. GO category overrepresentation analysis for reported as differential in the previous proteomics work, each two-way comparison. (XLSX 23 kb) such as the detoxifying enzymes already mentioned (redox Additional file 6: Table S4. Differential expression of cytoskeleton proteins and GSTs). related gene families. (XLSX 23 kb) Additional file 7: Table S5. Differential expression of collagen genes. Conclusions (XLSX 51 kb) In the first transcriptomic analysis of F. hepatica isolates Additional file 8: Table S6. Differential expression of redox genes. (XLSX 40 kb) with different levels of drug susceptibility, we were able to Additional file 9: Table S7. Differential expression of detoxifying highlight diverse protein functions and families that show enzymes. (XLSX 3441 kb) differential gene expression. Notably, several of the affected Additional file 10: Table S8. Differentially expression of genes genes and pathways correspond to those that are being identified in proteomic study. (XLSX 19 kb) proposed as normally altered upon drug exposure. The presence of variation in expression levels in these pathways Abbreviations ABZ: Albendazole; ABZR: Albendazole-resistant; ABZS: Albendazole-sensitive; in resistant isolates is suggestive, but we cannot conclude ABZ-SO: Albendazole sulphoxide; AC: Adenylate cyclase; DEG: Differentially with the available evidence that it is related to the resistant expressed genes; FDR: False discovery rate; FMO: Flavin mono-oxigenases; phenotype. Further experiments assessing expression levels GST: Gluthatione S-transferase; PGP: P-glycoprotein; PKA: Protein kinase A; TCBZ: Triclabendazole; TCBZ.SO: Triclabendazole sulphoxide; of these mRNAs in the different isolates upon controlled TCBZ.SO2: Triclabendazole sulphone; TCBZR: Triclabendazole-resistant; exposure are necessary to either confirm or reject that RNA TCBZS: Triclabendazole-sensitive levels actually vary upon drug exposure. While those experi- ments are on the way, we can highlight that the differentially Acknowledgments We are thankful for the support of agencies CABBIO, CAPES, ANII, PEDECIBA, expressed genes in resistant isolates are diverse, and and CSIC. The authors would like to thank Dr Martín Ciganda for correlate quite well with initial biochemical and structural proofreading the manuscript’s text and critical review of the results. observations of the pleiotropic nature of drug effects, Funding particularly in the case of TCBZ [17–24]. Moreover they The research was funded by grant CABBIO2012-UY04 awarded to JFT. also are coincident with more recent proteomic characterization of drug sensitive and resistant isolates from Availability of data and materials The datasets generated and analyzed during the current study are available European origins [46]. Interestingly a recent transcriptomic in the SRA repository, under accession PRJNA339158. study of drug-resistant isolates of Trypanosoma cruzi also showed altered expression of genes associated with putative Authors’ contributions SR performed most of the bioinformatic analysis and interpretation of data, and drug action mechanisms [81]. The fact that the parasites can contributed in writing the manuscript. SF contributed to the bioinformatic survivedrugexposurestronglysuggeststhatresistanceis analysis and the revision of manuscript content. VS, AMS, FMGA, PO, CH, EM, the result of additive subtle changes in the expression, and VG, FSMP, HS and GO participated in the data acquisition, contributed to conception of the study and were involved in the critical revision of the consequently protein metabolic activity. A corollary of this manuscript content. PS participated in the design of the study, contributed to observation is that resistance in different isolates might rely bioinformatic analysis and interpretation of data, and was a major contributor in Radio et al. Parasites & Vectors (2018) 11:56 Page 9 of 11

writing the manuscript. JT participated in the design of the study and the hatch test with isolates from South America and the United Kingdom. J interpretation of data, drafting the manuscript and critical revision of its Helminthol. 2014;88:286–92. content. All authors read and approved the final manuscript. 12. Mamani W, Condori R. Anthelminthic resistance (Fasciola hepatica) in sheep against albendazole and triclabendazole, La Paz - Bolivia. Rev Inv Vet Peru. Ethics approval and consent to participate 2009;20:254–62. The three laboratories that collected samples for this study followed protocols 13. Chavez A, Sanchez L, Arana C, Suarez F. Resistance to anthelmintics and approved by the respective local Committees of Animal Experimentation. prevalence of bovine fasciolosis in dairy farms in Jauja. Peru Rev Inv Vet Peru. 2012;23:90–7. Consent for publication 14. Gil LC, Díaz A, Rueda C, Martínez C, Castillo D, Apt W. 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